Despite decades of research, there is a fundamental gap in our understanding of how to adequately protect the intestines from the sequellae of ischemia/reperfusion (I/R) injury. We have accumulated multiple lines of evidence demonstrating that heparin-binding EGF-like growth factor (HB-EGF) can protect the intestines from I/R injury. The long-term goal of our work is to administer HB-EGF therapeutically to protect the intestines from injury. The overall objective of the current renewal application is to identify key elements of the interaction of HB- EGF with stem cells (SC) that may be utilized to augment the activity of the growth factor. Our central hypothesis is that a significant proportion of the intestinal cytoprotective effects of HB- EGF are mediated through its effects on SC, and that the interaction of HB-EGF with SC can be augmented in order to increase the efficacy of the growth factor. The rationale is that once the interactions of HB-EGF with SC are better understood, maximally beneficial innovative approaches to the treatment of intestinal I/R injury can be developed. We will objectively test our central hypothesis by pursuing the following specific aims: Aim 1) to determine the signaling mechanisms and receptors utilized by HB-EGF in protecting intestinal SC (ISC) from injury. We will use highly purified ISC to determine the effects of HB-EGF on the Wnt, BMP, PI3K and Notch signaling pathways in these cells. Aim 2) to determine whether endogenous HB-EGF expression affects ISC expansion and differentiation after injury. We will use a novel ex vivo crypt-villous organoid culture system and an in vivo intestinal I/R injury model to investigate the effects of HB-EGF on ISC expansion and differentiation. Aim 3) to determine whether bone marrow (BM)-derived SC can improve HB-EGF-mediated protection of the intestines from injury. We will determine the effects of SC mobilization or administration, in conjunction wth HB-EGF administration, in protection of the intestines from I/R injury in vivo. The results of our studies are expected to have an important positive impact in terms of providing improved therapeutic interventions for patients suffering from intestinal I/R injury, in addition to fundamentally advancing the understanding of growth factor/SC interactions in the intestines. This contribution is significant because it will enable us to best exploit the potential of clinical HB-EGF therapy. The proposed research is innovative because we seek to shift current experimental therapies for intestinal I/R injury away from the targeting of single inflammatory mediators, and towards strategies designed to protect and regenerate the intestinal mucosa, through administration of an intestinal cytoprotective growth factor in conjunction with multipotent SC.